Structural aspects of oligosaccharides have been studied by mass spectrometry (MS) for many years. The development of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization mass spectrometry (ESI-MS) for oligosaccharides have accelerated substantially the acceptance and utilization of MS-based technologies during the last decade. In the structural analysis of complex oligosaccharides originating from various isolated glycoproteins, MALDI-MS in conjunction with exoglycosidase digestion and a tandem MS/MS operation have become particularly popular.
MALDI-MS structural analysis of oligosaccharides can, in general, be conducted with native, i.e., underivatized, oligosaccharides. There are, however, several reasons for conversion of oligosaccharides into their permethylated derivatives. These reasons include a facile determination of i) branching, ii) interglycosidic linkages, and iii) the presence of configurational and conformational isomers. Permethylation also stabilizes sialic acid residues in acidic oligosaccharides, thereby yielding more predictable ion products when such oligosaccharides are subjected to MS/MS experiments, and permitting simultaneous analysis of neutral and sialylated oligosaccharides. Further, methylated sugars resulting from permethylation ionize more efficiently than their native, i.e., underivatized, counterparts. Permethylated oligosaccharides, being compatible with reversed-phase liquid chromatography (RPLC), also permit RPLC separation of permethylated oligosaccharides in complex oligosaccharide mixtures prior to MS analysis.
Current permethylation procedures, employed over a number of years in oligosaccharide analysis, are based on solution-phase methodologies. The currently more widespread solution-phase approach for permethylation is based on the addition of methyl iodide to oligosaccharides, which are dissolved in dimethyl sulfoxide (DMSO) containing powdered sodium hydroxide (NaOH). Although this solution-phase method is effective for replacing protons at oxygen and nitrogen sites in oligosaccharides, and has been used successfully in various MS structural studies of complex oligosaccharides, it is a multi-step procedure involving excessive, time-consuming sample handling steps and requiring excessive sample clean-up. Further, current solution-phase methods appear less satisfactory when low picomole to femtomole quantities of glycoprotein samples are available for permethylation, as is often the case when modern glycoanalysis of biological fluids and tissues is undertaken. This limitation is primarily due to oxidative degradation and “peeling,” i.e., side reactions, associated with the high pH resulting from dissolving NaOH powder prior to liquid-liquid extractions in solution-phase permethylation methods. These side reactions are adversely prominent with low picomole to femtomole quantities of glycoprotein samples.
Accordingly, permethylation procedures are needed that provide rapidity, experimental simplicity, clean reaction products (i.e., a low incidence of side reactions), scalability, and effective replacement of protons with methyl groups at oxygen and nitrogen sites in oligosaccharides.